CN101711775B - Fermentation composition for fermenting Agaricus blazei Murrill through probiotics - Google Patents
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Abstract
The invention relates to influence of probiotics on antineoplastic activity of polysaccharide of edible Agaricus blazei Murrill, in particular to a method for fermenting polysaccharide of Agaricus blazei Murrill through intestinal canal probiotics to increase and improve the antineoplastic activity of the polysaccharide of the Agaricus blazei Murrill. The invention further relates to a fermentation composition prepared by fermenting the polysaccharide extract of the Agaricus blazei Murrill through the intestinal canal probiotics, and an application of the fermentation composition in preparing health care products or medicines for preventing and curing tumor.
Description
Technical field
The present invention relates to the influence of probiotic bacterium, particularly relate to the Agaricus blazei polysaccharide and ferment, to improve and to improve the method for the anti-tumor activity of Agaricus blazei polysaccharide through beneficial bacteria of intestinal tract to the anti-tumor activity of Agaricus blazei polysaccharide.The invention further relates to the fermenting compsn that the Agaricus blazei polyoses extract makes after the beneficial bacteria of intestinal tract fermentation, and this fermenting compsn is used for preventing and treating the application of the healthcare products or the medicine of tumour in preparation.
Technical background
Agaricus blazei (Agaricus blazei Murrill) has another name called Agaricus blazei Murrill, is that the preciousness and rare that originates in Brazil is rich in sugar and proteinic dietotherapeutic fungi.There are some researches prove that Agaricus blazei has effects such as regulating immune, antitumor, antiviral, anti-inflammatory, antifatigue and protects the liver.
The anti-tumor activity of Agaricus blazei is from wherein contained polysaccharide peptide.Scholars such as Takashi Mizuno and Hagiwara are with hot water extraction from the Agaricus blazei sporophore, ethanol sedimentation, IX and affinity chromatography; Isolated polysaccharide is by 3 kinds of polysaccharose substances with powerful antitumor activity; Be FIO-a-α, FA-1-a-α and FA-1-a-β (Takashi Mizuno, Toshinikonagiqara, Antitumor activity and some properties of water-soluble polysacchrides from the fruiting body of Agaricus blazeiMurill; Agric.Biol.Chem.; 1990,54 (11), 2889-2996).
Someone thinks; Low-molecular-weight Agaricus blazei polysaccharide fraction has anti-tumor activity (Yoshiki Fujimiya et al.; Tumor-specific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the basidiomycete; Agaricus blazei Murill, Anticancer Research, 1999; 19:113-118), separate and from Agaricus blazei and be purified into 4 kinds of components such as ABM I-IV.Someone also proves, molecular weight is that 48000 daltonian ABM II is the strongest to spleen lymphocyte proliferation and K562 cytotoxic activity.For example, Chinese patent has been described for No. 200610151128.3 and has been used the zero(ppm) water lixiviate, the Agaricus blazei polysaccharide of the lower molecular weight (being about 48000 dalton) that ethanol sedimentation and chromatography method obtain, and the application in the preparation medicine for anti transfer of tumor.
Many experimental results of the prior art are all pointed out; The natural drug of known structure or unknown structure; After taking in the body through the gi tract approach, some structural change will take place, thereby can more easily be absorbed and the bigger BA of performance in human or animal body.Therefore among the present invention; We attempt to simulate normal people's intestinal microecology environment; Being utilized in distributes in the human body extensively and act on definite multiple beneficial bacteria of intestinal tract ferments with the single natural drug Agaricus blazei extract that the present invention uses, to realize the modification of beneficial bacteria of intestinal tract to Agaricus blazei.The result is surprisingly found out that, after handling with the beneficial bacteria of intestinal tract co-fermentation, the Agaricus blazei extract improves and improved its BA such as antitumor significantly.
Summary of the invention
The present invention relates to the influence of probiotic bacterium, particularly relate to the Agaricus blazei polysaccharide and ferment, to improve and to improve the method for the anti-tumor activity of Agaricus blazei polysaccharide through beneficial bacteria of intestinal tract to the anti-tumor activity of edible fungus polysaccharide.The invention further relates to the fermenting compsn that the Agaricus blazei polyoses extract makes after the beneficial bacteria of intestinal tract fermentation, and this fermenting compsn is used for preventing and treating the application of the healthcare products or the medicine of tumour in preparation.
Many researchs all confirm, and it is the highest that the anti-tumor activity of the contained polysaccharide of Agaricus blazei is that 15 kinds of glossy ganodermas etc. have in the edible mushrooms of anti-tumor activity an anti-tumor activity.The polysaccharide of Agaricus blazei, particularly low-molecular-weight polysaccharide (below 100,000 dalton) are the main active ingredient of its performance antitumor action.
An object of the present invention is to provide Agaricus blazei extract resulting tunning after probiotics fermention is handled.Among the present invention, the tunning that after probiotics fermention is handled, is obtained by the Agaricus blazei extract usually is called fermenting compsn for short.
According to the present invention, employed probiotic bacterium is selected from bifidus bacillus and probiotic lactobacillus in the Agaricus blazei fermentation.
According to a preferred embodiment of the invention, wherein be used to the to ferment said bifidus bacillus of Agaricus blazei is selected from bifidumbacterium bifidum, JCM 1192T, bifidobacterium adolescentis and bifidobacteria infantis; Wherein said probiotic lactobacillus is selected from Lactobacterium acidophilum, lactobacillus delbruckii, lactobacterium casei and lactobacillus rhamnosus.
According to a preferred embodiment of the invention, wherein be used to the to ferment said bifidus bacillus of Agaricus blazei preferably is selected from bifidumbacterium bifidum and JCM 1192T; Wherein said probiotic lactobacillus preferably is selected from lactobacterium casei and lactobacillus delbruckii.
Another object of the present invention provides the method for the above-mentioned fermenting compsn of preparation, and this method comprises utilizes superfine communication technique, pulverizes the Agaricus blazei cell and makes its cell wall breaking, farthest obtains the effective constituent of Agaricus blazei.With the Agaricus blazei polysaccharide that extracts or directly with the bacterium powder of broken wall Agaricus blazei as fermentation substrate, inoculate pre-incubated each about 0.5% (volume) one or both or two or more bifidus bacillus bacterium liquid.The conventional cultivation after about 2～6 hours inoculated one or both or two or more probiotic lactobacillus bacterium liquid of pre-incubated each about 0.5% (volume) once more, continues fermentation 20～22 hours.Reduce to 4.0 when following when the pH value, finish fermentation, promptly obtain the fermenting compsn (referring to embodiment 1 and 2) of fermenting Agaricus blazei Murrill through probiotics.
Generally speaking, during the fermenting compsn of preparation fermenting Agaricus blazei Murrill through probiotics, the Agaricus blazei consumption is to contain Brazilian mushroom powder 2-20 gram in every 100ml culture.
Usually adopt superfine communication technique to prepare the Brazilian mushroom powder of cell wall breaking.In the past, use common breaking method to prepare the powder of the diffusing state of rough segmentation mostly, the recovery of effective constituent is often lower like this, and bioavailability is also lower.In order in high volume production process, to obtain effective constituent to greatest extent, the Chinese medicine preparation production field has been introduced superfine communication technique.Agaricus blazei epigranular after handling through micronizing, fine and closely woven, and cell walls also is broken, thereby increased the specific surface area of bacterium powder, thus more help the stripping of effective ingredient, increased the medicinal material extraction ratio of effective constituents greatly.Broken wall Brazil mushroom powder smashing fineness of micronizing is more than 100 orders.
Can in by the bacterium powder that the broken wall Agaricus blazei constitutes, add and be equivalent to the long-pending about 30 times pure water of Agaricus blazei bacterium powder, and soak 30 minutes.Extracted about 1 hour down in 100 ℃ then.After the filtration, in residue, add about 30 times water again, extracted 1 hour down in 100 ℃ once more.After the filtration, collect filtrating, merge extracted twice liquid, and be evaporated to wherein that the concentration of Agaricus blazei is 20% (weight/volume), promptly obtain required Agaricus blazei polysaccharide extraction liquid.
In order to prepare the Agaricus blazei polysaccharide substrate that is used for probiotics fermention, can directly utilize the Brazilian mushroom powder of broken wall, consumption is to contain Brazilian mushroom powder 2-20 gram in every 100ml culture, and then adds glucose 0.5% (weight/volume), inorganic salt (ferrous sulfate 7H
2O 1.0mg/100ml, zinc sulfate 7H
2O 4.4mg/100ml, Sodium Selenite 5H
2O 0.024mg/100ml, sal epsom 7H
2O 70mg/100ml, manganous sulfate H
2O0.5mg/100ml, sodium-chlor 1.0mg/100ml, potassium primary phosphate 100mg/100ml, potassium hydrogenphosphate 3H
2O 100mg/100ml), about adjustment pH7.0 and 115 ℃ of sterilizations after 40 minutes, promptly obtain continue after be used for the Agaricus blazei polysaccharide substrate of probiotics fermention.
The Agaricus blazei polysaccharide extraction liquid that aforesaid method capable of using extracts, as the probiotics fermention culture substrate, about adjustment pH7.0 and 115 ℃ of sterilizations after 40 minutes, promptly obtain continue after be used for the Agaricus blazei polysaccharide substrate of probiotics fermention.
After the Agaricus blazei polysaccharide substrate sterilization that is used for the probiotic bacterium transformation fermentation of preparation as stated above; When temperature is reduced to 39 ℃ of 2 left and right sides; Inoculate the bacterium liquid of a kind of or two kinds of bifidus bacilluss of pre-incubated each about 0.5% (volume); The conventional cultivation after about 2～6 hours inoculated the bacterium liquid of a kind of or two kinds of probiotic lactobacillus of pre-incubated each about 0.5% (volume) once more, continues fermentation 20～22 hours.The pH value is reduced to 4.0 and is finished fermentation when following, promptly obtains the fermenting compsn (referring to embodiment 1 and 2) of fermenting Agaricus blazei Murrill through probiotics.
In order to prove that probiotics fermention handles, Agaricus blazei is to the influence of probiotic bacterium growth, our experimental observation after probiotics fermention handles Agaricus blazei, the variation of probiotic bacterium number.Test-results shows that after together fermenting with Agaricus blazei, the thalline number of probiotic bacterium is not less than the probiotic bacterium quantity (referring to embodiment 3) of growing in the conventional substratum of MRS.
In order to disclose the probiotics fermention chemically modified effect possible to the Agaricus blazei molecule, our fundamental research experiment confirms that also after probiotics fermention was handled, the limiting viscosity of Agaricus blazei substrate solution [η] obviously reduced.This results suggest is after probiotics fermention is handled, because the cracking of Agaricus blazei polysaccharide molecule cause the polysaccharide molecule number significantly to increase, and the molecular-weight average of polysaccharide reduces (referring to embodiment 4).
Another purpose of invention provides fermenting compsn of the present invention and in the medicine of enhance immunity power, prevention and treatment tumour or healthcare products, uses.
Be fermenting compsn that detects fermenting Agaricus blazei Murrill through probiotics of the present invention and the tumor killing effect that does not pass through the Agaricus blazei polysaccharide of probiotic bacterium transformation fermentation, two test samples directly come into operation in carrying experimental S
180The animal tumor model of sarcoma.Agaricus blazei polysaccharide through probiotics fermention transforms all has the obvious suppression effect to tumour, and high, medium and low three dose groups are respectively 53.71%, 38.2% and 0.26% to the tumour inhibiting rate of mouse S180.And the Agaricus blazei polysaccharide that transforms without probiotics fermention, high, medium and low three dose groups are respectively 38.90%, 0.69% ,-0.37% to the tumour inhibiting rate of mouse S180.It is thus clear that, through after the probiotic bacterium transformation fermentation, the tumor-inhibiting action of Agaricus blazei polysaccharide be significantly improved (specifically seeing embodiment 5).Though relevant mechanism is still not clear; But this result of study proves; Agaricus blazei polysaccharide or other fungus polysaccharides are under intestinal beneficial bacterium crowd's interaction in vitro; Through after some known or unknown chemically modified or bio-transformation, be more conducive to the absorption of human body utilization, brought into play higher biological function (referring to embodiment 5).
Learn and the interior animal experiment confirmation through cell in vitro, the antigen peptide of modification of the present invention can promote the propagation of T lymphocyte and mammary cancer specific CTL effectively, excites the killing activity of immunity system to oncocyte, thereby reaches the purpose that suppresses tumor growth.Particularly our comparative experiments shows, compares with wild-type sequence, and the antigen peptide that the present invention modifies can more effectively promote the propagation of T cell, improves the ability of T emiocytosis IFN-γ, thereby has improved the killing activity of CTL to tumour cell.
Though the present invention is that example explanation Agaricus blazei is after probiotics fermention is handled with the Agaricus blazei; Significantly improved the BA of Agaricus blazei; Its anti-tumor activity particularly; But it will be appreciated by those skilled in the art that probiotics fermention is handled and is applicable to that equally also fermentative processing comprises other fungies of needle mushroom, mushroom, glossy ganoderma etc.
Embodiment
Embodiment 1: the preparation of the fermenting compsn of fermenting Agaricus blazei Murrill through probiotics polysaccharide
Get the exsiccant Agaricus blazei, adopt cell wall breaking technology to be crushed to breaking trachytectum (Brazilian mushroom powder smashing fineness is more than 100 orders).In Brazilian mushroom powder, add the pure water that is equivalent to long-pending 30 times of bacterium powder and soaked 30 minutes, and extract 1 hour after-filtration in 100 ℃.In filter residue, add 30 times water again, 100 ℃ were extracted 1 hour, after filtering once more, merged extracted twice liquid, and to be evaporated to the concentration that contains Agaricus blazei be 20% (weight/volume), were the Agaricus blazei polysaccharide extraction liquid.With 115 ℃ of heating sterilizations in 40 minutes down of Agaricus blazei polysaccharide extraction liquid.When temperature is reduced to 39 ℃ of left and right sides, insert preparatory cultured bifidumbacterium bifidum and JCM 1192T liquid, inoculum size is 0.5% (volume).Ferment after 6 hours, insert pre-incubated lactobacillus delbruckii, each 0.5% (volume) of lactobacterium casei bacterium liquid again.The fermentation culture temperature is about 37 ℃, continues fermentation 20～22 hours.The pH value reduces to 4.0 when following, finishes fermentation, promptly obtains the fermenting compsn of fermenting Agaricus blazei Murrill through probiotics polysaccharide.
Embodiment 2: the preparation of the fermenting compsn of fermenting Agaricus blazei Murrill through probiotics bacterium powder
Get the exsiccant Agaricus blazei, adopt cell wall breaking technology, pulverize, sediments microscope inspection, spore is broken wall.Require the Agaricus blazei powder can pass through 100 mesh sieves; Consumption is to contain Brazilian mushroom powder 2 grams in every 100ml culture; Add glucose 0.5% (weight/volume), inorganic salt (ferrous sulfate 7H2O 1.0mg/100ml, zinc sulfate 7H2O 4.4mg/100ml, Sodium Selenite 5H2O 0.024mg/100ml, sal epsom 7H2O70mg/100ml, manganous sulfate H2O 0.5mg/100ml, sodium-chlor 1.0mg/100ml, potassium primary phosphate 100mg/100ml, potassium hydrogenphosphate 3H2O 100mg/100ml) again, the adjustment pH7.0 about and 115 ℃ the sterilization 40 minutes.When temperature is reduced to 39 ℃ of left and right sides, insert preparatory cultured bifidumbacterium bifidum and JCM 1192T bacterium liquid, inoculum size is 0.5% (volume).Ferment and insert pre-incubated lactobacillus delbruckii and lactobacterium casei bacterium liquid after 6 hours again, inoculum size is 0.5% (volume).37 ℃ are continued fermentation 20～22 hours, reduce to 4.0 when following when pH value, and end is fermented, and obtains the fermenting compsn of fermenting Agaricus blazei Murrill through probiotics bacterium powder.
Embodiment 3: the fermentation growing state of probiotic bacterium in Agaricus blazei polysaccharide extraction liquid and Agaricus blazei bacterium foundation cream thing
In the substratum according to embodiment 1 and 2 preparations, inoculation bifidumbacterium bifidum and JCM 1192T bacterium liquid (inoculum size is ditto said) fermented after 6 hours, and sampling detects zymophyte thalline number.And then insert pre-incubated lactobacillus delbruckii and lactobacterium casei bacterium liquid (inoculum size is ditto said), continue to be cultured to 48 hours.The sampling in the 24th, 32 and 48 hour of wherein fermenting detects the bacterium number.According to same operation steps inoculation MRS substratum, as contrast.Detection is carried out according to the method described in " check of microbiological test of food hygiene bifidus bacillus " GB/T4789.34-2003.Detected result is as shown in table 1.
Table 1: the growth bacterium number of probiotic bacterium in the Agaricus blazei substratum
Can find out that from table 1 with conventional MRS substratum relatively, human body probiotic bifidobacteria, probiotic lactobacillus are well-grown in the environment of substratum at Agaricus blazei polysaccharide and Brazilian mushroom powder, all can reach 100,000,000 (10 of every milliliter of mycetome amounts
8) more than.And, fermentation growth after 24 hours the bacterium number reach the highest, as continue to cultivate then that the thalline number reduces gradually.This experiment shows, probiotic bacterium can katabolism Agaricus blazei polysaccharide in process of growth, obtains growth preferably.Continue to cultivate, because the accumulation and the nutraceutical minimizing of metabolite, number of viable constantly reduces.
Embodiment 4: viscosimetry detects the variation of Agaricus blazei polysaccharide probiotics fermention front and back molecular-weight average
The Agaricus blazei polysaccharide is a kind of fungus polysaccharide with good resistance tumor promotion, and through behind the probiotics fermention, its tumor-inhibiting action obviously improves.The physico-chemical property of polysaccharide especially physiologically active is relevant with distribution with its molecular weight.For this reason, the inventor utilizes viscosimetry, has measured the RELATIVE MOLECULAR WEIGHT OF PLASTIC of probiotics fermention front and back Agaricus blazei polysaccharide.According to test, in enough rare solution: after polysaccharide, solvent, temperature etc. are confirmed, [η] (being called limiting viscosity) value relevant with the relative molecular mass M of superpolymer.Try to achieve inherent viscosity [η] with semiempirical Mike's nonlinear equation:
[η]＝KM
α
In the formula: the MV of M----polysaccharide relative molecular mass;
The K----rate constant;
α----and the relevant experience of the form of superpolymer in solution.
Prepare the sample solution of 5 kinds of different concns, measure the relative viscosity η of each concentration
r, utilize sulfuric acid-phynol method to detect the content of Agaricus blazei polysaccharide.The Agaricus blazei polysaccharide sample of fermentation is an original content, and the Brazilian mushroom powder fermented liquid of fermentation concentrates 10 times, is contrast with unfermentable Agaricus blazei polysaccharide.Three test result of samples such as tables 2.η
r-1/c maps to concentration C, with straight-line extrapolation, with the intersection point of Y axle be [η].Shown in the following tabulation 2 of result.
Table 2: three sample detection results of Agaricus blazei polysaccharide
[η] can find out from the limiting viscosity shown in the table 2, and Agaricus blazei polysaccharide and Brazilian mushroom powder are through behind the probiotics fermention, and its numerical value obviously reduces.According to formula [η]=KM
α, [η] value is big more, and the relative molecular weight M of polysaccharide is just big more.Therefore can infer that the Agaricus blazei polysaccharide is through behind the probiotics fermention, molecule is caused its relative molecular weight obviously to reduce by cracking.
Embodiment 5: the fermenting compsn of fermenting Agaricus blazei Murrill through probiotics polysaccharide and unfermentable Agaricus blazei polysaccharide anti-tumor activity are relatively
Present embodiment utilizes animal model for tumour, describes the Agaricus blazei polyoses extract for example and is learning active improvement through the external fermentation conversion of beneficial bacteria of intestinal tract artifact.
Get the Agaricus blazei polysaccharide extraction liquid sample of embodiment 1 preparation, be divided into two parts.Portion carries out probiotics fermention, and another part do not ferment.After fermentation is accomplished, two duplicate samples are evaporated to the half the of original volume.Use sulfuric acid phynol method (Food science, 2004,25 volumes, 7 phases, " mensuration of Agaricus Blazei Murrill polysaccharide ") to measure the fermentation and the polysaccharide content of fermented sample not then.The result is visible, and in the Agaricus blazei polysaccharide fermentation sample, polysaccharide content is 35.1mg/ml, and the polysaccharide content of fermented sample is not 31.8mg/ml.
The equal body weight of making even is that 70 Kunming kind small white mouses of 18-22g are divided 7 groups, 10 every group at random.The 1st group is saline water group (blank), and animal is only accepted saline water 0.6ml; 2nd, 3,4, group is the Agaricus blazei large, medium and small dose groups of fermented sample not, dosage is respectively 0.8g/kg, 0.4g/kg and 0.2g/kg; 5th, 6,7 groups is the large, medium and small dose groups of Agaricus blazei fermented sample, and dosage is respectively 0.8g/kg, 0.4g/kg and 0.2g/kg.Every day gastric infusion once, successive administration 7 days.The 8th day, get the solid tumor piece that the tumor animal well-grown does not have diabrosis under the aseptic condition, be cut into fine grained chippings with scissors, in knurl piece (g): saline water (ml) is 1: 3 ratio, grinds to form homogenate with glass homogenizer, counts the oncocyte number in every milliliter of slurries.Give the inoculation 2 * 10 down of every mouse armpit
6Cell suspension 0.2ml, inoculation continued administration 10 days.Disconnected neck was put to death animal on 1, and it is heavy with knurl to weigh, and calculates inhibition rate of tumor growth (%) by following formula.
In the experiment of this treatment of animals, tumour inhibiting rate is regarded as by test agent said tumour being had antitumor (or tumor suppression) activity greater than 30%, and tumour inhibiting rate then is regarded as not having antitumor (or tumor suppression) activity less than 30%.
Every batch of laboratory animal requires same sex (hero).Shown in the following tabulation 3 of experimental result.
Table 3: Agaricus blazei Murrill is to tumor-inhibiting action result in mouse S180 (solid tumor) body
Annotate: with negative control group than * P＜0.05 * * P＜0.01
From the result of this test, can find out; After the Agaricus blazei polysaccharide transforms through probiotics fermention; Its each dose groups tumor-inhibiting action is apparently higher than not fermentation group, infer maybe the Agaricus blazei polysaccharide through the effect of probiotic bacterium, the enzyme that probiotic bacterium produces with the polysaccharide long-chain of the macromolecule of Agaricus blazei Murrill be hydrolyzed to micromolecular, have a more polysaccharide fragment of powerful antitumor activity; Thereby increased the activeconstituents of performance tumor-inhibiting action in the Agaricus blazei polysaccharide, improved the tumor-inhibiting action of Agaricus blazei polysaccharide.
Claims (2)
1. fermentation culture compsn that is used to prevent and treat the fermenting Agaricus blazei Murrill through probiotics of tumour; It is characterized in that this fermentation culture compsn be to use the Agaricus blazei polyoses extract, or directly utilize the Brazilian mushroom powder of broken wall, according to the preparation of following method; Said method comprises utilizes superfine communication technique to pulverize Agaricus blazei to breaking trachytectum, uses 100 ℃ of hot water extraction Agaricus blazei polysaccharide then, and is fermentation substrate with the Agaricus blazei polysaccharide that extracts; Perhaps directly utilize the Brazilian mushroom powder of broken wall to be fermentation substrate; Inoculate the bifidumbacterium bifidum of pre-incubated each 0.5% (volume) and the bifidus bacillus bacterium liquid of JCM 1192T, the conventional cultivation after about 2～6 hours, and then inoculate the lactobacillus delbruckii of pre-incubated each 0.5% (volume) and the probiotic lactobacillus bacterium liquid of lactobacterium casei; 37 ℃ are continued fermentation 20～22 hours; The pH value reduces to 4.0 when following, finishes fermentation, promptly obtains the fermentation culture compsn of fermenting Agaricus blazei Murrill through probiotics.
2. be used for preventing and the medicine or healthcare products of treating tumour are used in production according to the fermentation culture compsn of claim 1.
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